Growth hormone (GH) and prolactin (PRL) interact with GH and prolactin receptors (GHR; PRLR), both cytokine receptors, causing activation of the GHR- and PRLR-associated tyrosine kinase, JAK2, and the STATS and ERK pathways. Much is known about how GH and PRL directly affect gene expression and cell behavior. Less is known about how they interact with other growth factors to modify signaling and biological responses when cells are exposed to GH or PRL along with the growth factor - a situation more like in vivo. This proposal addresses two aspects of "crosstalk" between: 1) GH or PRL and epidermal growth factor (EGF) and 2) GH and insulin-like growth factor-1 (IGF-1). In preadipocytes, we find that GH, acting via ERKs, causes EGF receptor (EGFR) threonine phosphorylation, retards EGF-induced EGFR downregulation, and enhances EGF-induced signaling. Preliminary results reveal similar crosstalk between PRL and EGF in T47D human breast cancer cells. We also find that GH promotes association of IGF-1 receptor (IGF-1 R) with the GHR-JAK2 complex and that IGF-1 enhances GH-induced signaling. GH-IGF-1 crosstalk is not ERK-dependent, but may reflect IGF-1-induced changes in GHR activatability. We hypothesize: 1) GH and PRL, in addition to direct signaling, exert indirect effects via alteration of cellular EGF sensitivity by promoting EGFR threonine phosphorylation and influencing EGF-activated EGFR's intracellular itinerary. 2) GH collaborates with IGF-1 by using common signaling elements, thus broadening the idea that GH and IGF-1 exert independent and overlapping actions. Our aims are: 1. Define mechanisms by which GH and PRL, via ERK activation, influence EGF signaling, EGFR trafficking, and biological effects. We will test GH's effects on EGFR's association/subcellular colocalization with proteins that mediate its downregulation. By reconstituting EGFR-deficient cells, an EGFR threonine phosphorylation mutant's impact on EGFR downregulation and GH-EGF crosstalk will be analyzed. We will study the role of EGFR threonine phosphorylation in PRL-EGF crosstalk in vivo by monitoring T47D cell tumor behavior with bioluminescence imaging. 2. Investigate the basis and significance of physical and functional interaction of GH and IGF-1 signaling elements. The role of JAK2 in GH-induced GHR-JAK2- IGF-1R complex formation will be determined and regions of GHR required will be mapped. The role of IGF-1 R kinase activity in allowing GH-IGF-1 signaling synergy will be tested. GH-IGF-1 crosstalk will be studied in human prostate cancer cells and murine osteoblasts. These studies will significantly expand knowledge of GH and cytokine signaling and of biologicallyrelevant crosstalk between cytokine and growth factor signaling systems.